Abstract: Pseudomonas aeruginosa, a plant growth-promoting rhizobacterium and Memnoniella echinata, a fungal antagonist used separately or in combination in soil amended with neem cake and/or chemical fertilizers significantly suppressed root infecting fungi viz., Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani and the root-knot nematode Meloidogyne javanica in mungbean. Soil amendment increased biocontrol and growth promoting potential of P. aeruginosa and M. echinata.
Introduction
Fluorescent Pseudomonads are a group of bacteria that have long been found in the rhizosphere of a wide range of plants (Curl and Truelove, 1986). Many isolates from this group are known to demonstrate antagonistic activity against other micro-organisms of the soil and therefore, have been intensively used in trials for the biological control of soil-borne plant diseases. Memnoniella echinata (Riv.) Golloway, a cosmopolitan dematiaceous fungus is well known as a decomposer of cellulosic material (Ellis, 1971) has been reported to inhibit growth of root-infecting fungi in vitro (Abbas and Ghaffar, 1973) and in green house conditions (Siddiqui et al., 1998). Oil cakes are generally rich in mineral ingridients as nitrogen, phosphorus and potash (Akhtar, 1991). Use of oil cakes of castor (Mehta et al., 1994), ground nut (Ali and Mien, 1989) and neem (Goswami, 1993; Rathore, 1994) has shown good control of plant parasitic nematodes. An experiment was therefore carried out to evaluate the effects of M. echinata and Pseudomonas aeruginosa, a plant growth-promoting rhizobacterium (Siddiqui et al., 1999b) in soil with or without amendment of soil with neem cake and/or chemical fertilizers for the control of soil-borne root-infecting fungi viz., Macrophomina phaseolina, Rhizoctonia solani and Fusarium solani and the root-knot nematode Meloidogyne javanica in mungbean.
Materials and Methods
The soil used for the experiment was a sandy-loam of pH 8.1 obtained from the experimental field of the Department of Botany, University of Karachi. The soil had a natural population of 6-8 sclerotia of M. phaseolina as estimated by wet sieving and dilution technique (Sheikh and Ghaffar, 1975), 4.5% colonization of R. solani on sorghum seeds used as baits (Wilhelm, 1955) and 3000 cfu g–1 of soil of mixed population of Fusarium spp., as determined by soil dilution technique (Nash and Snyder, 1962). Powdered neem cake mixed in soil, at 1% w/w were transferred in 8-cm diam., plastic pots at 350 gm in each. In another set urea (at 0.15 g/kg soil) and potash (at 0.12 g/kg) with or without neem cake were used. The pots were watered daily and kept at 50% W.H.C. (Keen and Raczkowski, 1921). After three weeks, a 25 ml conidial or cell suspension of M. echinata at 2.1×108 cfu/ml and P. aeruginosa at 2.5x108 cfu/ml were drenched in soil. Eight seeds of mungbean (Vigna radiata (L.) Wilczek) were sown in each pot. Each treatment was replicated three times and the pots were randomized on a screen house bench. After germination only 4 seedlings were maintained in each pot. After one week of the seedling emergence, 2000 eggs/J2 of M. javanica were inoculated near the root zone by making three holes around mungbean roots. Observations on root infection caused by root-knot nematode and root-infecting fungi were recorded. To determine the incidence of root-infecting fungi, roots after washing in running tap water were surface disinfested with 1% Ca(OCl)2 and one cm long root pieces from tap root were transferred on to PDA plates containing penicillin (100,000 units/l.) and streptomycin sulphate (0.2 g/l). The dishes were incubated for 5 days at 28oC to confirm infection and colonization of roots by soil-borne root infecting fungi. Infection percentage was calculated as follows:
Plant growth parameters in terms of plant height, shoot weight, root weight and number of Rhizobium-nodules per plant were also recorded. Data were analyzed and subjected to one way analysis of variance (ANOVA) followed by Standard Error of the Difference between means (SED) according to Gomez and Gomez (1984).
Results
Treatment showed significant difference on the development of root-knot infection in mungbean. Maximum suppression in root-knot development was achieved in the treatment where P. aeruginosa was used in combination with neem cake and urea followed by with neem cake-potash P. aeruginosa treatment which resulted in >49 percent inhibition in root-knot disease (Table 1). Treatments showed non-significant difference in the suppression of M. phaseolina, F. solani and R. solani infections. More than 50 percent inhibition in M. phaseolina infection was observed in the treatment where neem cake used alone, P. aeruginosa used with M. echinata, P. aeruginosa mixed with potash, M. echinata mixed with either neem cake, urea or potash, P. aeruginosa either mixed with neem cake and urea or neem cake and potash and M. echinata either mixed with neem cake and urea or neem cake and potash. Similarly, neem cake, urea and P. aeruginosa used individually, P. aeruginosa and M. echinata used together, P. aeruginosa used with urea, M. echinata used with either neem cake or urea and a P. aeruginosa-neem cake-potash treatment showed more than 50 percent inhibition in F. solani infection. Almost all the treatments suppressed R. solani infection by more than 50 percent (Table 1).
Neem cake used with potash produced maximum plant height whereas a neem cake-urea M. echinata treatment resulted in maximum fresh weight of shoot.
| Table 1: | Effect of neem cake and chemical fertilizers on the efficacy of Pseudomonas aeruginosa and Memnoniella echinata on the development of root-knot and root-rot infection in mungbean |
| Table 2: | Effect of neem cake and chemical fertilizers on the efficacy of Pseudomonas aeruginosa and Memnoniella echinata on the development of root-knot and root-rot infection in mungbean |
Similarly, a neem cake-potash P. aeruginosa treatment produced greatest fresh weight of roots. Urea mixed with M. echinata produced highest number of Bradyrhizobium-nodules per root system (Table 2).
Discussion
The nutrition of a plant determines in large measure its resistance or susceptibility to disease, its histological or morphological structure or properties, the function of tissues to hasten or slow pathogenesis, and the virulence and ability of pathogens to survive. Non availability of nutrient elements needed to synthesize chemical and physical barriers or the diversion of elements into metabolic cul-des-sacs around infection sits, can result in susceptability to disease (Huber, 1980). In the present study, P. aeruginosa and M. echinata used in soil amended with neem cake and/or chemical fertilizers effectively suppressed root-infecting fungi and the root-knot nematode. Chemical fertilizers have been reported to suppress root-rot and root-knot diseases in mungbean (Siddiqui et al., 1999a). Potassium is also known to reduce Fusarium oxysporum infection on tomato (Eliot, 1973) and Rhizoctonia solani infection on hemp (Pal and Chaudhary, 1980). Use of potash has shown reduction in M. javanica population (Oteifa, 1953). The deficiency of potash increases root exudation (Marschner, 1986), hence attract more micro-organisms in the rhizosphere. The mechanism involved in the control of plant-parasitic nematodes by urea was described by Huebner et al. (1983) where urea when added to soil is finally converted into ammonia gas and high concentration of ammonia have shown nematicidal effect in killing nematodes (Rodriguez-Kabana et al., 1987). There are reports where neem cake and neem oil used as seed treatment were found effective in reducing root penetration by M. incognita juveniles in mungbean (Vijayalakshmi and Goswami, 1986). Reduced hatching and inhibited penetration by M. incognita was also observed by Mojumder and Mishra (1991) when chickpea seeds were soaked in neem cake and Brassica rapa extracts.
In the present study, P. aeruginosa and M. echinata used alone or in combination showed significant suppression in root-knot diseases. P. aeruginosa has been reported to control root-rot and root-knot diseases in chilli (Siddiqui et al., 1999b) and tomato (Siddiqui et al., 1999c). Similarly, root infection caused by M. phaseolina, F. solani and R. solani was significantly suppressed following application with M. echinata (Siddiqui et al., 1998).
It is interesting to note that biocontrol agents used in soil amended with neem cake and/or chemical fertilizers showed better results as compared to their separate use. Addition of organic matter to soil stimulates the microbial activity of bacteria, fungi, algae and other micro-organisms (Rodriguez-Kabana et al., 1987).
The result of the present study would therefore suggest that P. aeruginosa and M. echinata with a compatible amendment could be use in the suppression of soil-borne root-infecting fungi and plant-parasitic nematodes which may results in better plant growth.